in vivo 25: 307-314 (2011)
Xenotropic Murine Leukemia Virus-related Virus-associated Chronic Fatigue Syndrome Reveals a Distinct Inflammatory Signature
VINCENT C. LOMBARDI1, KATHRYN S. HAGEN1, KENNETH W. HUNTER4, JOHN W. DIAMOND2, JULIE SMITH-GAGEN3, WEI YANG3 and JUDY A. MIKOVITS1
1Whittemore Peterson Institute, University of Nevada, Reno MS 0552, 1664 N. Virginia St., Reno, NV 89557, U.S.A.;
2Triad Medical Center, 4600 Kietzke Lane M242,Reno, NV 89502, U.S.A.;
3Nevada Center for Health Statistics and Informatics, University of Nevada, 1664 N. Virginia St., Reno, NV
89557, U.S.A.;
4University of Nevada Reno, Department of Microbiology and Immunology Applied Research Facility,1664 N. Virginia St., MS 199, Reno, NV 89557 U.S.A.
Abstract.
Background:
The recent identification of xenotropic murine leukemia virus-related virus (XMRV) in the blood of patients with chronic fatigue syndrome (CFS) establishes that a retrovirus may play a role in the pathology in this disease.
Knowledge of the immune response might lead to a better understanding of the role XMRV plays in this syndrome.
Our objective was to investigate the cytokine and chemokine response in XMRV-associated CFS.
Materials and Methods:
Using Luminex multi-analyte profiling technology, we measured cytokine and chemokine values in the plasma of XMRV-infected CFS patients and compared these data to those of healthy controls.
Analysis was performed using the Gene Expression Pattern Analysis Suite and the Random Forest tree classification algorithm.
Results:
This study identifies a signature of 10 cytokines and chemokines which correctly identifies XMRV/CFS patients with 93% specificity and 96% sensitivity.
Conclusion:
These data show, for the first time, an immunological pattern associated with XMRV/CFS.
````
Introduction
Chronic fatigue syndrome (CFS) is a poorly understood disease of unknown etiology, which is commonly characterized by innate immune defects, chronic immune activation and dysregulation, often leading to neurological maladies [reviewed in (1)].
It can also involve other biological systems such as the musculoskeletal, gastrointestinal and endocrinological systems (2-4).
Although several common symptoms are primarily reported and predominate, they may differ among individuals, are often intermittent and can persist for years, frequently resulting in substantial disability
(5).
Some of the most commonly reported physical symptoms include muscle weakness and pain, tender or swollen lymph nodes and chronic flu-like symptoms (6).
Memory and concentration impairment, blurred vision, dizziness and sleep abnormalities represent some of the cognitive symptoms typically observed while immunological symptoms often manifest themselves through viral reactivation, RNase L dysregulation, decreased natural killer (NK) cell function and susceptibility to opportunistic infections (7-12).
NK cell dysregulation may be associated with viral reactivation or viral persistence and may also lead to malignancy (13, 14).
Indeed, clinical observations corroborate pathological manifestations in CFS as viral reactivations, particularly herpes virus such as cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpes virus-6 (HHV-6), are common occurrences (15-17).
Moreover, epidemiological studies have reported increased incidences of lymphoma associated with CFS outbreaks (18 ).
These clinical observations suggest that a compromised innate immune system may play a role in CFS pathology.
The completion of the human genome project enabled positional cloning studies to identify the RNASEL gene as the hereditary prostate cancer allele-1 (HPC1) (19).
This discovery prompted Robert Silverman and his colleagues to search for a viral component to hereditary prostate cancer.
Using a viral micro-array and tissue biopsies from individuals with hereditary prostate cancer they identified and sequenced the complete genome of a novel human gammaretrovirus, very similar in sequence to xenotropic murine leukemia virus and therefore termed the new virus xenotropic murine leukemia virus-related virus (XMRV) (20).
Subsequent studies performed in our laboratory identified and isolated infectious XMRV in the blood of 67% of CFS patients (21).
This work was performed using multiple techniques including PCR, electron microscopy showing budding viral particles, Western blot analysis of viral proteins and serology confirming that infected patients express antibodies to XMRV envelope proteins.
In addition, gene sequencing and phylogenetic analysis confirmed these patients were indeed infected with XMRV that was >99% identical to previously published sequences but was obviously distinct from the only existing XMRV molecular clone, VP62 (20).
Taken together, this work clearly rules out any possibility of gross contamination and additionally, represents the first identification and isolation of naturally occurring infectious XMRV.
The connection between CFS and XMRV was further supported by the studies of Lo et al., who identified murine leukemia virus (MLV)-related sequences in the blood of 86% of CFS patients, further establishing a retroviral association with CFS (22).
Presently, three families of retroviruses are known to infect humans; the human immunodeficiency viruses
(HIV), the human T-cell leukemia viruses (HTLV) and now the human murine leukemia-related viruses.
Both HIV and HTLV are known to dysregulate the innate immune system and promote the production of inflammatory cytokines and chemokines (23, 24).
In light of the association between XMRV and CFS, it is not surprising that some of the most salient observations in CFS are the differences in cytokines and chemokines when compared to healthy controls (8 ).
Previous reports, however, addressing the role of
these molecules in CFS have produced conflicting
results.
Much of this emerges from such hindrances as small sample size, a limited number of cytokines surveyed at one time, insufficient patient population stratification, and insufficient negative control subjects.
This has resulted in inconsistent reports in the literature for a number of cytokines including interleukins (IL) 6, 10 and 12.
In spite of these conflicting results, a number of cytokines and chemokines have consistently been show to be associated with different subgroups of CFS.
For instance, Natelson et al. showed elevated levels of IL-8 and IL-10 in the cerebral spinal fluid of patients with sudden, influenza-like onset CFS when compared to healthy controls (25).
Additionally, Chao et al. have show neopterin and IL-6 to be up-regulated in subsets of CFS patients, indicative of a pro-inflammatory immune condition (26).
However, these studies did not analyze the complex relationships between multiple cytokines and clinical disease.
By applying conventional statistical analysis and 'machine logic' algorithms to the multiplex data, it is possible to identify cytokines and chemokines that are differentially expressed between two groups.
To support this premise, we have used the xMap multi-analyte profiling technology that allows simultaneous measurements of multiple biomarkers in serum or plasma.
In this study, a panel of 26 cytokines, chemokines and growth and angiogenic factors were analyzed in blood plasma of CFS patients and healthy control subjects.
This study revealed a signature of 10 cytokines and chemokines, which showed a specificity of 93% and sensitivity of 96% in diagnosing XMRV-associated CFS in this patient cohort.
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Xenotropic Murine Leukemia Virus-related Virus-associated Chronic Fatigue Syndrome Reveals a Distinct Inflammatory Signature
VINCENT C. LOMBARDI1, KATHRYN S. HAGEN1, KENNETH W. HUNTER4, JOHN W. DIAMOND2, JULIE SMITH-GAGEN3, WEI YANG3 and JUDY A. MIKOVITS1
1Whittemore Peterson Institute, University of Nevada, Reno MS 0552, 1664 N. Virginia St., Reno, NV 89557, U.S.A.;
2Triad Medical Center, 4600 Kietzke Lane M242,Reno, NV 89502, U.S.A.;
3Nevada Center for Health Statistics and Informatics, University of Nevada, 1664 N. Virginia St., Reno, NV
89557, U.S.A.;
4University of Nevada Reno, Department of Microbiology and Immunology Applied Research Facility,1664 N. Virginia St., MS 199, Reno, NV 89557 U.S.A.
Abstract.
Background:
The recent identification of xenotropic murine leukemia virus-related virus (XMRV) in the blood of patients with chronic fatigue syndrome (CFS) establishes that a retrovirus may play a role in the pathology in this disease.
Knowledge of the immune response might lead to a better understanding of the role XMRV plays in this syndrome.
Our objective was to investigate the cytokine and chemokine response in XMRV-associated CFS.
Materials and Methods:
Using Luminex multi-analyte profiling technology, we measured cytokine and chemokine values in the plasma of XMRV-infected CFS patients and compared these data to those of healthy controls.
Analysis was performed using the Gene Expression Pattern Analysis Suite and the Random Forest tree classification algorithm.
Results:
This study identifies a signature of 10 cytokines and chemokines which correctly identifies XMRV/CFS patients with 93% specificity and 96% sensitivity.
Conclusion:
These data show, for the first time, an immunological pattern associated with XMRV/CFS.
````
Introduction
Chronic fatigue syndrome (CFS) is a poorly understood disease of unknown etiology, which is commonly characterized by innate immune defects, chronic immune activation and dysregulation, often leading to neurological maladies [reviewed in (1)].
It can also involve other biological systems such as the musculoskeletal, gastrointestinal and endocrinological systems (2-4).
Although several common symptoms are primarily reported and predominate, they may differ among individuals, are often intermittent and can persist for years, frequently resulting in substantial disability
(5).
Some of the most commonly reported physical symptoms include muscle weakness and pain, tender or swollen lymph nodes and chronic flu-like symptoms (6).
Memory and concentration impairment, blurred vision, dizziness and sleep abnormalities represent some of the cognitive symptoms typically observed while immunological symptoms often manifest themselves through viral reactivation, RNase L dysregulation, decreased natural killer (NK) cell function and susceptibility to opportunistic infections (7-12).
NK cell dysregulation may be associated with viral reactivation or viral persistence and may also lead to malignancy (13, 14).
Indeed, clinical observations corroborate pathological manifestations in CFS as viral reactivations, particularly herpes virus such as cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpes virus-6 (HHV-6), are common occurrences (15-17).
Moreover, epidemiological studies have reported increased incidences of lymphoma associated with CFS outbreaks (18 ).
These clinical observations suggest that a compromised innate immune system may play a role in CFS pathology.
The completion of the human genome project enabled positional cloning studies to identify the RNASEL gene as the hereditary prostate cancer allele-1 (HPC1) (19).
This discovery prompted Robert Silverman and his colleagues to search for a viral component to hereditary prostate cancer.
Using a viral micro-array and tissue biopsies from individuals with hereditary prostate cancer they identified and sequenced the complete genome of a novel human gammaretrovirus, very similar in sequence to xenotropic murine leukemia virus and therefore termed the new virus xenotropic murine leukemia virus-related virus (XMRV) (20).
Subsequent studies performed in our laboratory identified and isolated infectious XMRV in the blood of 67% of CFS patients (21).
This work was performed using multiple techniques including PCR, electron microscopy showing budding viral particles, Western blot analysis of viral proteins and serology confirming that infected patients express antibodies to XMRV envelope proteins.
In addition, gene sequencing and phylogenetic analysis confirmed these patients were indeed infected with XMRV that was >99% identical to previously published sequences but was obviously distinct from the only existing XMRV molecular clone, VP62 (20).
Taken together, this work clearly rules out any possibility of gross contamination and additionally, represents the first identification and isolation of naturally occurring infectious XMRV.
The connection between CFS and XMRV was further supported by the studies of Lo et al., who identified murine leukemia virus (MLV)-related sequences in the blood of 86% of CFS patients, further establishing a retroviral association with CFS (22).
Presently, three families of retroviruses are known to infect humans; the human immunodeficiency viruses
(HIV), the human T-cell leukemia viruses (HTLV) and now the human murine leukemia-related viruses.
Both HIV and HTLV are known to dysregulate the innate immune system and promote the production of inflammatory cytokines and chemokines (23, 24).
In light of the association between XMRV and CFS, it is not surprising that some of the most salient observations in CFS are the differences in cytokines and chemokines when compared to healthy controls (8 ).
Previous reports, however, addressing the role of
these molecules in CFS have produced conflicting
results.
Much of this emerges from such hindrances as small sample size, a limited number of cytokines surveyed at one time, insufficient patient population stratification, and insufficient negative control subjects.
This has resulted in inconsistent reports in the literature for a number of cytokines including interleukins (IL) 6, 10 and 12.
In spite of these conflicting results, a number of cytokines and chemokines have consistently been show to be associated with different subgroups of CFS.
For instance, Natelson et al. showed elevated levels of IL-8 and IL-10 in the cerebral spinal fluid of patients with sudden, influenza-like onset CFS when compared to healthy controls (25).
Additionally, Chao et al. have show neopterin and IL-6 to be up-regulated in subsets of CFS patients, indicative of a pro-inflammatory immune condition (26).
However, these studies did not analyze the complex relationships between multiple cytokines and clinical disease.
By applying conventional statistical analysis and 'machine logic' algorithms to the multiplex data, it is possible to identify cytokines and chemokines that are differentially expressed between two groups.
To support this premise, we have used the xMap multi-analyte profiling technology that allows simultaneous measurements of multiple biomarkers in serum or plasma.
In this study, a panel of 26 cytokines, chemokines and growth and angiogenic factors were analyzed in blood plasma of CFS patients and healthy control subjects.
This study revealed a signature of 10 cytokines and chemokines, which showed a specificity of 93% and sensitivity of 96% in diagnosing XMRV-associated CFS in this patient cohort.
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